The Leishmania genome project reference strain, Leishmania major Friedlin, is trisomic for chromosome 1. The complete sequence of this chromosome has revealed that the genes are grouped into two large clusters of the polycistronic type, each borne by one DNA strand and located on each side of a 1.6-kb sequence often termed the switch region. Several hypotheses concerning the role of this switch region have been put forward (region of initiation of transcription for both gene clusters, origin of replication or centromeric sequence). In the present study, we have deleted this region on the three copies of chromosome 1 by sequential targeted replacements. The absence of the switch region did not alter the mitotic stability of the three deleted chromosomes. This region therefore does not appear necessary for chromosomal replication or segregation. However, during the third targeting round, which aimed at knocking out the last switch region, a fourth copy of chromosome 1 that retained this region appeared in all clones analysed. This suggests that the persistence of this switch region is necessary for parasite survival. We then showed that the presence/absence of the switch region did not act upon the expression of a resistance marker gene inserted beforehand into the left gene cluster of the same chromosomal molecule. This result suggests that the presence of this 1.6-kb sequence is not necessary for the expression of all genes on chromosome 1.